Maternal behavior changes after immune challenge of neonates with developmental effects on adult social behavior

To examine whether maternal responsiveness during interactions with endotoxin-treated pups contributes to long-term effects on social development, neonatal mice were fostered on postnatal day 1 to dams from three selectively bred lines that differ in social behaviors. On day 5, neonates were administered saline or 0.5 mg/kg endotoxin (lipopolysaccharide, i.p.). Observations of undisturbed dams and litters on days 2, 4, 6, and 8 showed modest line differences in maternal behaviors. At the peak intensity of the transient illness induced by endotoxin (3 hr postinjection on day 5), dams increased licking and decreased time off-nest for endotoxin, but not saline-treated pups. As adults, fostered-reared males were observed in brief social interactions. Males exposed to endotoxin early in life showed changes in adult social behaviors that depended on foster dam line as well as individual differences in maternal responsiveness. Maternal responsiveness to stressed neonates can ameliorate the social-developmental effects of early illness.     

Mild prenatal stress in rats is associated with enhanced conditioned fear

We tested the hypothesis that prenatal stress would enhance conditioned fear in adult rats. Pregnant Sprague-Dawley rats were stressed by exposure to a novel environment and subcutaneous injection of saline (0.1 ml 0.9% NaCl) at random times daily from Days 14 to 21 of pregnancy. When compared to adult control (CON) male rats from unmanipulated pregnancies, adult prenatally stressed (PS) male rats showed increased freezing behavior in response to acute footshock as well as increased freezing behavior the next day in the same context, without shock delivery. In another experiment, the gestational stressor was examined for elevations in corticosterone and ACTH. At gestational days (G)15, G17, G19 and G21, maternal and fetal plasma was collected. Analysis showed elevations in corticosterone and ACTH in the PS dams when compared to the CON dams. Additionally, increased corticosterone was found in the PS fetuses when compared to the CON fetuses. Finally, some CON and PS litters were examined for alterations in length of gestation, number of pups born, bodyweight on postnatal day (P)1 and anogenital distance on P1 and differences were not found. In conclusion, our data demonstrate that a mild stressor during gestation, sufficient to raise plasma corticosterone and ACTH, is associated with enhanced conditioned fear during adulthood.     

Sirenomelia: analysis in the cadmium- and lead-treated golden hamster

Sirenomelia, a fusion of the lower extremities, is believed to result from a median, bilateral symmetric defect of the caudal portion of the embryo at a very early stage in development. Anomalies of the gastrointestinal and urogenital systems are commonly associated with this malformation. Sirenomelia is not an embryo-lethal condition but typically is incompatible with postnatal life when combined with the associated malformations. In this study, intravenous treatment of hamsters with a combination of cadmium and lead on the morning of the eighth day of gestation resulted in 1.4, 22.2, and 35.6% of the viable fetuses displaying sirenomelia in litters recovered on days 15, 12, and 10 of gestation, respectively. With the exception of several fetuses with exencephaly, most structures cranial to the level of the umbilicus were normal. Caudal abdominal and pelvic structures were severely affected, with agenesis or dysgenesis of the kidneys, bladder, umbilical arteries, and external genitalia frequently noted. The administration of a combination of cadmium and lead has proven to be an effective and consistent means of inducing sirenomelia in the golden hamster.     

Transplacental transmission of polyoma virus in mice.

When pregnant mice were inoculated on day 1 of gestation with polyoma, some of them exhibited total resorption or reduced litter size, the extent depending on the dose of virus. Virus was detected in 4 out of 11 mouse embryo fibroblast (MEF) cultures made from infected mothers. After maternal infection on day 5 or 10 of gestation, virus titers of up to 10(7) 50% tissue culture infectious doses (TCID50)/g of fetus were found in all pools of fetuses tested 5 days later, with the titers falling by day 6. Hemagglutination-inhibiting antibodies against polyoma appeared in maternal serum by day 6 and rose to a maximum by day 14. Immunoglobulin G class antibodies were detected by day 7, with titers rising rapidly to a maximum at day 14. After maternal infection later in gestation (day 15), one out of three litters of newborn mice was found to have 10(5) TCID50 polyoma virus per g in pooled kidney samples.     

Effects of induced thyroid deficiency on the development of suckling behavior in rats

The effects of propylthiouracil (PTU) on the ontogeny of suckling behavior in rats were examined. The drug was given at two dosage levels of 0.3% and 0.5% respectively mixed with rat diet throughout gestation and suckling. The thyroid glands of treated fetuses and pups and of untreated control animals of the same age groups were monitored by histologic examination. At the behavioral level, the frequency of individual movements of head, forelimbs and mouth was significantly reduced in treated fetuses. The combination movements of head, mouth and forelimbs showed severe deficits both quantitatively and qualitatively for all ages in the experimental group from day 18 of gestation. Hypothyroid pups of dams raised on 0.5% PTU were unable to attach to the nipple of the mother and died within a few days. Pups of dams raised on 0.3% PTU showed longer latencies for nipple attachment, and their gross motor movements of rooting and suckling were greatly impaired. These results have been discussed in relation to the development of suckling behavior to indicate that, during ontogeny, some decisive step in the integration of individual movements takes place in utero from day 18 of gestation. This coincides with the establishment of pituitary thyroid relationship, which is continued through postnatal stages.     

Effect of valproic acid on fetal and maternal organs in the mouse: a morphological study

Valproic acid (VPA) is an antiepileptic drug used clinically. Because of its known teratogenic properties VPA is not recommended for women of child bearing age. The present study was designed to assess the effects of VPA on both fetal and maternal organs. Randomized groups of pregnant mice were treated as follows: Group 1 (n = 10) 500 mg/kg VPA/day on gestation days 8-11; Group 2 (n = 10) 600 mg/kg VPA/day on gestation days 8-11; and Group 3 (n = 4) saline-injected controls. On gestation day 18, the pregnant mice were euthanized, fetuses collected and prepared for scanning electron microscopy. In addition, fetal and maternal organs were processed for routine histology, immunohistochemistry for growth factors (TGF alpha, beta-1, beta-2 and EGF) and transmission electron microscopy. Scanning microscopy revealed specific lesions induced by VPA in the fetus, namely spina bifida occulta, exencephaly, and exophthalmia. On the other hand, there were no detectable morphological changes in fetal or maternal organs by routine histology, immunohistochemistry or electron microscopy. The data suggest that the lesions present in the fetus are due to a direct effect by VPA on retinoic acid, a ubiquitous compound that has a role in normal development, rather than the lack of transport of sufficient nutrients to the fetus as a result of placental insufficiency due to VPA-induced toxicity.     

Valproic acid-induced fetal malformations are reduced by maternal immune stimulation with granulocyte-macrophage colony-stimulating factor or interferon-gamma

Valproic acid, a drug commonly used to treat seizures and other psychiatric disorders, causes neural tube defects (NTDs) in exposed fetuses at a rate 20 times higher than in the general population. Failure of the neural tube to close during development results in exencephaly or anencephaly, as well as spina bifida. In mice, nonspecific activation of the maternal immune system can reduce fetal abnormalities caused by diverse etiologies, including diabetes-induced NTDs. We hypothesized that nonspecific activation of the maternal immune system with interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) could reduce valproic acid (VA)-induced defects as well. Female CD-1 mice were given immune stimulant prebreeding: either IFN-gamma or GM-CSF. Approximately half of the control and immune-stimulated pregnant females were then exposed to 500 mg/kg VA on the morning of gestational day 8. The incidence of developmental defects was determined on gestational day 17 from at least eight litters in each of the following treatment groups: control, VA only, IFN-gamma only, IFN-gamma+VA, GM-CSF only, and GM-CSF+VA. The incidence of NTDs was 18% in fetuses exposed to VA alone, compared to 3.7% and 2.9% in fetuses exposed to IFN-gamma+VA, or GM-CSF+VA respectively. Ocular defects were also significantly reduced from 28.0% in VA exposed groups to 9.8% in IFN-gamma+VA and 12.5% in GM-CSF+VA groups. The mechanisms by which maternal immune stimulation prevents birth defects remain unclear, but may involve maternal or fetal production of cytokines or growth factors which protect the fetus from the dysregulatory effects of teratogens.     

Genetic damage detected in CD-1 mouse pups exposed perinatally to 3'-azido-3'-deoxythymidine or dideoxyinosine via maternal dosing, nursing, and direct gavage: II. Effects of the individual agents compared to combination treatment

We previously reported extraordinary increases in micronucleated erythrocytes in CD-1 mouse pups exposed to 3'-azido-3'-deoxythymidine (AZT) and dideoxyinosine (ddI; 50/250, 75/375, 150/750 mg/kg/day AZT/ddI) by gavage throughout gestation and lactation, followed by direct pup dosing beginning postnatal day (PND) 4 (Bishop et al. [2004]: Environ Mol Mutagen 43: 3-9). That study was conducted to explore the potential for genetic damage in newborns exposed perinatally to antiretrovirals in order to reduce maternal-infant transmission of HIV-1. Because dramatic increases in frequencies of micronucleated erythrocytes were seen in exposed pups, additional studies were conducted to clarify the relative contribution of each drug to the observed damage. Pregnant CD-1 mice were administered AZT (50, 75, 150 mg/kg/day) or ddI (250, 375, 750 mg/kg/day) by gavage twice daily in equal fractions beginning prior to mating and continuing throughout gestation and lactation. Direct pup dosing (same regimens) began on PND 4. Peripheral blood erythrocytes of male pups were screened for micronuclei on PNDs 1, 4, 8, and 21. Significant increases in micronucleated erythrocytes were observed in pups and dams exposed to AZT at all doses and sampling times. The highest micronucleus levels were observed in pups on PND 8 after the initiation of direct dosing. In contrast, effects seen in pups and dams treated with ddI were minimal. These results demonstrate that AZT, a component of many anti-HIV combination therapies, induces chromosomal damage in perinatally exposed neonatal mice. Comparison of micronucleated cell frequencies induced by AZT alone or in combination with ddI suggests that ddI potentiates AZT-induced chromosomal damage following direct exposure.     

Infection with equine herpesvirus 1 (EHV-1) strain HVS25A in pregnant mice

The abortigenic effects of equine herpesvirus 1 (EHV-1) strain HVS25A, given intranasally, were assessed in pregnant BALB/c, C57BL/6J and Quakenbush mice at day 16 of pregnancy. All EHV-1-infected BALB/c mice showed clinical signs typical of EHV-1-induced disease, together with evidence of abortion. However, although there were fetal and neonatal deaths in some C57BL/6J and Quakenbush litters, the respiratory and systemic effects of EHV-1 infection in the dams were inconsistent. BALB/c dams were then inoculated at day 15 of pregnancy with either EHV-1 or rabbit kidney (RK) cell lysate (controls) and animals were killed at days 1-5 post-inoculation (pi), i.e., before the occurrence of abortions. EHV-1-infected mice showed a significant fall in rectal temperature between days 1 and 2 pi and lost weight during the first 4 days pi, demonstrating a significant mean difference in weight gain from the control group at days 2, 3, 4 and 5 pi. Death in utero was seen in five of 90 fetuses of EHV-1-infected mice, but in no fetuses from RK-inoculated mice. On days 1, 2, 3, 4 and 5 pi, the fetuses from EHV-1-infected dams were significantly smaller than those from RK-inoculated dams. Congestion and necrosis of the middle layer of trophoblast and chorionic necrosis were observed in the placentae from EHV-1-infected dams and assessed by a scoring system. Virus was isolated rarely from the fetuses (1/73), placentae (3/72) and uteri (1/16) of EHV-1-infected dams, and only from those killed on day 1 or 2 pi. This indicates that, as in the horse, abortion caused by EHV-1 infection in mice is not necessarily a consequence of fetal infection but may be due to fetal compromise due to vascular effects on the placenta.     

A cross-fostering analysis of the effects of PCB 77 on the maternal behavior of rats

Polychlorinated biphenyls (PCBs) are environmental contaminants known to cause multiple behavioral and developmental problems in humans and animals. In rats, gestational exposure to the PCB congener 3, 4, 3', 4'-tetrachlorobiphenyl (PCB 77) affects the brain and behavior of the offspring as well as the maternal behavior of the dams. Whether the behavior of dams is affected by direct effects of the contaminant or indirectly by actions of the PCB on the developing offspring is not known. We investigated this question using a cross-fostering paradigm in which pregnant rats were exposed to either oil vehicle or 2 mg/kg of PCB 77 on gestational days 6 through 18, and then raised pups that had been exposed to either oil or PCB 77 during gestation. Maternal behavior was observed on postnatal days 1, 2, 4 and 6. Some of the effects on maternal behavior, including an increase in the frequency of nursing bouts and in the amount of maternal autogrooming, can be ascribed to prenatal exposure of the litters to the PCB. Other behavioral effects, including an increase in time on the nest and in the amount of pup grooming as well as a reduction in high-crouch nursing, appear to be due to both direct effects of the PCB on the dams and effects mediated by changes in the offspring. Our results show that exposure to PCB 77 can have complex effects on the behavioral interactions between the dams and their litters with a potential impact on the development of the offspring.     

Developmental toxicity of dinocap in the mouse is not due to two isomers of the major active ingredients

Technical-grade dinocap, a complex-mixture fungicide, is teratogenic in the CD-1 mouse, causing cleft palate and otolith defects. In this study we compared the developmental toxicities of 2,4-dinitro-6-(1-methylheptyl)phenyl crotonate and 2,6-dinitro-4-(1-methylheptyl)phenyl crotonate, model isomers of the major active ingredients of technical dinocap, to the known teratogenicity of the technical compound. Individual isomers, both isomers combined, or technical dinocap were administered to pregnant mice on days 7-16 of gestation. Some dams were killed at term and litters were removed, dead fetuses and resorptions were counted, and live fetuses were weighed and preserved in Bodian's fixative for examination for cleft palate. Other treated dams were allowed to give birth: postnatal viability and growth, development of swimming behavior, and otolith formation were evaluated. As in previous studies, technical-grade dinocap caused cleft palate and weight deficits in fetuses at term and increased neonatal mortality and abnormal swimming behavior, torticollis, and deficient otolith formation in surviving pups. Neither of the purified isomers exhibited any developmental toxicity when administered under identical conditions. Thus, it is concluded that these isomers are not the active teratogenic component(s) in technical-grade dinocap.     

Effects of acute hypergravity exposure and parity on maternal behavior in CD-1 mice

We assessed the behavioral response to acute hypergravity exposure in lactating mother mice, Mus musculus of the outbred CD-1 strain. Primiparous or terziparous dams were exposed with their litters to a centrifugal force equivalent to 2G hypergravity for 1 h daily from postnatal day 2 (P2) to P9. We made detailed behavioral observations before, during and after the rotation on selected days to identify elements of the maternal behavioral repertoire vulnerable to 2G challenge. Licking and nest building were reduced during rotation while mothers sniffed and snouted their pups more. Nursing and total time in physical contact with pups were relatively stable. The effects of rotation were most pronounced on P2, dams appearing to habituate to the treatment with repeated exposure. Dam parity had a limited effect on the behavioral response to rotation, primiparous mothers tending to spend longer nursing their pups during the rotation and showing a greater tendency to lick and nest-build in post-rotation. Differences between parity groups diminished over days. Body weight was decreased in rotated primiparous dams and their pups gained less weight than stationary controls. Ultrasonic vocalization (USV) rates recorded on P2, P5 and P9 seemed to indicate delayed behavioral development in rotated pups.     

Mouse hepatitis virus and host determinants of vertical transmission and maternally-derived passive immunity in mice

Summary Transmission of mouse hepatitis virus (MHV) in utero following oronasal inoculation of pregnant mice was found to depend upon MHV strain and host genotype. Virulent, polytropic MHV-JHM was recovered from multiple maternal tissues, including liver and uterus, as well as placenta and fetus in susceptible BALB/cByJ mice. Fetuses were infected during all 3 trimesters of pregnancy. Low virulence, polytropic MHV-S infected fetuses in a low percentage of susceptible BALB/cByJ dams. Infection of resistant CD-1 mice with MHV-JHM was limited, with no fetal infection. Enterotropic MHV-Y was largely restricted to intestine of BALB/cByJ and CD-1 dams, with minimal dissemination and no fetal infection. Maternally-derived MHV IgG antibody was detectable in pup sera through 4 weeks of age. Antibody titers were generally lower in second litters of the same dam. Cross-fostering experiments showed that antibody was transferred via colostrum and not in utero, and that pups were capable of absorption through 2 weeks of age. Pups nursing immune dams were protected against MHV challenge at 1 and 2 weeks of age, compared to pups nursing naive dams. Immunity to MHV challenge was cross-protective against both antigenically homotypic and heterotypic strains of MHV.     

Sodium intake and reproduction in BALB/C mice.

The effect of sodium intake on the reproductive performance of BALB/C mice was assessed in four groups of 11 or 12 mice that received ad lib access to low or higher sodium food (LSF 4-5, HSF 120-143 mmol Na+/kg). The two groups that received HSF had (mean values) 100% matings, 83 and 91% litters, 5.9 pups/litter, pups weighing 2.05 and 2.22 g (3 days after birth) and 10.47 and 10.96 g at weaning (19 days). One of the HSF groups that also had 300 mM NaCl to drink did not show any benefit. Two groups received LSF, and one of them also received 30 mM NaCl. The group given LSF only had 83% matings, 20% litters, 1.5 pups/litter, and pups that were significantly smaller at birth and at weaning. However, the LSF group given 30 mM NaCl to drink performed almost as well as the two HSF groups. The results show that (a) the daily sodium requirement for optimal reproduction was > or = 400 (micromol/day, based on voluntary sodium intake late in gestation and lactation; (b) sodium deficiency was the cause of reproductive deficiency in mice on LSF; (c) severe sodium deficiency suppressed reproduction primarily at the gestation step; (d) this deficiency could be prevented by the voluntary sodium intake of mothers with access to salt solution; and (e) pups on the LSF showed an avid innate salt appetite when offered salt solution at 12 days of age.     

Effect of exogenous thyroxine on the development of the Purkinje cell in fetal alcohol effects in the rat

The amelioration of fetal alcohol effects on the postnatal development of the Purkinje cell by exogenous L-thyroxine was investigated in the neonatal rat. Time-pregnant rats were divided into three groups. Group A (n = 6) received 35% liquid ethanol diet; Group B (n = 6) was fed a liquid diet in which maltose dextrins replaced alcohol isocalorically, constituting the pair-fed group; Group C (n = 6) received the 35% liquid ethanol diet and, in addition, received exogenous thyroxine (5 microg/kg/day) subcutaneously. After the pups were born, the mothers were removed and the pups of each were surrogate fostered by dams who were fed normal rat chow and water ad libitum. An average of six pups, one from each litter, were killed at days 7, 14, 21, and 28 for each of the above three groups. Light and electron microscopic observations of lobule II/III revealed a delayed alignment of Purkinje cells (Pc) in alcohol-exposed pups compared to pair-fed pups. The Pc of the pair-fed group showed a single-layer arrangement at 7 days which was seen only at day 14 in the alcohol group. However, in the alcohol + T(4)-exposed pups a single-layer arrangement was quite often seen at 7 days. Morphological observations showed impaired evidence of protein synthesis at all time sequences in the pups of Group A compared to Group B. A most interesting finding was the morphological evidence of greater protein synthesis in the Pc of the alcohol + T(4) group at all times as indicated by a hypertrophied nucleus, abundant ribosomal collection, and numerous Nissl bodies.     

Radiation-induced quantitative alterations in prenatal thymic development in the beagle dog

Quantitative morphology of the canine fetal thymus was studied to evaluate the age-dependent radiosensitivity of the developing immune system. Pregnant beagle dams received abdominal 60Co gamma exposures (200 R) or were sham irradiated at one of three ages in gestation, 30, 40, or 45 days. The mean calculated dose to each fetus was 1.5 Gray. One-half of the fetuses in each litter were harvested by hysterotomy at 5 days and one-half at 10 days post-irradiation (PI). The volumes of the thymic lobules and lobular cortices were significantly reduced at 5 and 10 days PI when compared with age-matched controls. Thymic cortical volumes in irradiated fetuses were reduced between 13 and 29% from control volumes by 5 days PI and 8 and 13% by 10 day PI. Thymic medullary volumes in irradiated fetuses were reduced 18 to 23% by 5 days PI and 27 to 54% by 10 days PI. The reductions in medullary volumes in fetuses irradiated at 35, 40, and 45 days of gestation and evaluated at 10 days PI were 54, 38, and 27%, respectively. Although injury to both thymic cortices and medullas was greater following exposures earlier in gestation, damage to medullas was relatively more severe than in cortices following exposure at any one age. The degree of reduction of medullary volume reflects thymic epithelial injury and is surprising since thymic epithelium is considered to be radioresistant in the adult. Such injury may have serious consequences postnatally as normal differentiation of T cell subpopulations is dependent upon the integrity of the thymic microenvironment. Damage to the thymic microenvironment could result in defects in immunologic regulation and in immune deficiencies.     

Fertility,maternal care,and offspring behavior in mice prenatally treated with tobacco smoke

Female mice from lines selectivity bred for differences in open-field activity were exposed to tobacco smoke during gestation. Smoke-treated females were less likely than controls to have produced litters by 23 days after observation of a vaginal plug. Within the high-active line, fewer pups of smoke-treated dams survived to weaning. Regardless of treatment, fewer high-active than low-active offspring survived to weaning. Results of a 4-day series of open-field activity tests administered to offspring beginning at 28 days of age indicated that tobacco smoke administered prenatally and/or during testing depresses open-field activity in both lines. Other activity tests administered at 50 days of age gave similar results. Tissue nicotine levels after nicotine injection tended to be higher in high-active and control groups than in low-active and smoke-treated groups, respectively. Liver weight expressed as percentage of body weight was 11.9% greater in smoke-treated animals than in controls.     

Maternal diesel inhalation increases airway hyperreactivity in ozone-exposed offspring

Air pollutant exposure is linked with childhood asthma incidence and exacerbations, and maternal exposure to airborne pollutants during pregnancy increases airway hyperreactivity (AHR) in offspring. To determine if exposure to diesel exhaust (DE) during pregnancy worsened postnatal ozone-induced AHR, timed pregnant C57BL/6 mice were exposed to DE (0.5 or 2.0 mg/m(3)) 4 hours daily from Gestation Day 9-17, or received twice-weekly oropharyngeal aspirations of the collected DE particles (DEPs). Placentas and fetal lungs were harvested on Gestation Day 18 for cytokine analysis. In other litters, pups born to dams exposed to air or DE, or to dams treated with aspirated diesel particles, were exposed to filtered air or 1 ppm ozone beginning the day after birth, for 3 hours per day, 3 days per week for 4 weeks. Additional pups were monitored after a 4-week recovery period. Diesel inhalation or aspiration during pregnancy increased levels of placental and fetal lung cytokines. There were no significant effects on airway leukocytes, but prenatal diesel augmented ozone-induced elevations of bronchoalveolar lavage cytokines at 4 weeks. Mice born to the high-concentration diesel-exposed dams had worse ozone-induced AHR, which persisted in the 4-week recovery animals. Prenatal diesel exposure combined with postnatal ozone exposure also worsened secondary alveolar crest development. We conclude that maternal inhalation of DE in pregnancy provokes a fetal inflammatory response that, combined with postnatal ozone exposure, impairs alveolar development, and causes a more severe and long-lasting AHR to ozone exposure.     

Models of infantile malnutrition in rats: effects on maternal behavior

The effects of 6 commonly used methods for producing malnutrition in suckling rats upon the behavior of their dams were examined. In these methods, (1) large litters were fostered on the dams; (2) dams were fed low-protein diets; (3) dams were fed an inadequate amount of a good quality diet; (4) pups were removed from the dams and kept with a nonlactating female for half of the day; (5) pups were removed from the dams and kept in an incubator for half of the day; or (6) some of th dams' teats were destroyed. Dams that were themselves food deprived showed a shift from resting to exploratory behaviors with some indication of increased attention to the pups compared to controls. Dams whose pups were starved but that were well fed themselves showed lower levels of resting and higher levels of attention to pups but no elevation in exploratory activity. Although the long-term significance of the behavioral changes of the dams is not known, the immediate effect was to minimize the impact of the experimenter's manipulations on the metabolic energy available to the pups for growth and development.     

Effects of mother-litter separation on later maternal responsiveness in the hamster

Primiparous hamsters were permitted either 1,24, or 48 hr of contact with their own litters beginning at parturition followed by varying periods of separation from pups. A different subgroup in each of these 3 conditions was tested for maternal behavior each day starting 24 hr after litter removal. Testing for the display of retrieving and the adoption of the nursing posture using 1–2 day old foster pups continued for each condition until 2 subgroups failed to behave maternally on 2 consecutive days. Nulliparous females were similarly exposed to foster pups and tested at selected intervals; regardless of initial exposure, only 4 of 24 nulliparous animals ultimately displayed maternal behavior. Different primiparous subgroups given 1 hr of exposure were maternal on Days 2–4 postpartum but not on Days 5 or 6 while females in the 24 hr group were maternal on Days 3–5 but not on Days 7 or 8. Groups permitted 48 hr of litter stimulation were maternal through Day 15 postpartum demonstrating that the initial 24–48 hr of pup contact is essential for the development of a long-term mother-young bond.